The purpose of this proposal is to identify a temporal pattern of specific genetic alterations in the nucleotide excision repair (NER) pathway that are associated with a multistep carcinogenic progression of normal to premalignant to invasive breast cancer in BRCAl-mutated breast epithelial cells. The long-term objective is to identify specific molecular markers that are predictive of breast cancer risk, and that may serve as targets for prevention or early detection of breast cancer in this population. Key components of this project are to utilize MRI screen-detected breast tissue and premalignant cells identified in ductal lavage specimens in BRCA1 carriers to address the hypothesis. The aim are as follows: Aim 1: To evaluate nucleotide excision repair activity and sensitivity to DNA damaging agents in a BRCAI-null human cell line. I have previously shown that overexpression of BRCA1 enhances NER of ultraviolet (UV)-induced photoproducts and that a potential mechanism is through transcriptional regulation of NER genes. These experiments were done in cell lines that contain endogenous BRCA1. I will confirm these results in a BRCAI-null human cell line and extend the analysis to determine cellular susceptibility to different DNA damaging agents that are targeted by NER for repair and are more relevant for breast cancer treatment. Aim 2: To determine if MRI screen-detected breast lesions in BRCA1 carriers contain additional genetic mutations or alterations in the expression of nucleotide excision repair DNA-damage recognition genes compared to normal tissues from these individuals. I will analyze this tissue for LOH of the normal allele of BRCA1, mutations in the p53 gene, and alterations in the NER genes that are regulated by BRCA1 and p53. Aim 3: To determine the expression levels of nucleotide excision repair DNA-damage-recognition genes and evaluate nucleotide excision repair activity in benign (histologically normal or hyperplastic), atypical, and malignant ductal cells from women with BRCA1 mutations. LOH of BRCA1, mutations in p53, and loss of expression of NER genes will be correlated with histopathology. In order to validate the functional consequences of these alterations, assays for repair activity will be performed on these ductal cells.